Abstract
A method free from methodological artefacts is essential for determining the thermal inactivation kinetics of microorganisms. In this work, the stirred flask method was adopted to examine the thermal inactivation kinetics of exponential and stationary-phase S. Typhimurium cells at three different temperatures: 55°C, 57°C, 60°C, using aerobic and anaerobic enumeration methods. Survivor curves obtained in this way showed that stationary-phase cells were more heat resistant than exponential-phase cells, while the use of anaerobic enumeration methods did not enhance cell recovery. Exponential phase cells were significantly more heat resistant at 55°C when heated in a stationary-phase cell culture (ca. 109 cfu/ml) previously inactivated for 40-min at the investigation temperature. Exponential cell cultures heated for the same period before reinoculation were not protective. The protection afforded by the stationary-phase culture was a combined effect of high cell density (over 107 cfu/ml) and period of heat-treatment (no longer than 40-min) lowering the redox potential of the medium. The shift in redox potential was shown to be correlated with the dissolved oxygen concentration during heat-treatment so that protection of exponential-phase cells against thermal death could be the result of protection against oxidative damage during heat inactivation. The addition of other oxidants and reductants showed that the measured heat resistance of exponential Salmonella cells was independent of the oxygen concentration and was solely dependent on the redox potential. The results further showed that pre-reduction of heating media could increase the thermal resistance of exponential cells of Salmonella even if not coupled with anaerobic recovery conditions. The instantaneous protection afforded by the heated stationary-phase Salmonella cultures was not a result of a component present in the culture able to initiate adaptive gene expression. Using a real time assay for functional RpoS that relied on bioluminescence as a reporter, it was shown that RpoS had no role in mediating the protective effect via the stationary-phase adaptive response. The time required for RpoS induction in S. Typhimurium (pSB367) at 37°C in a previously heated for 40-min at 55°C stationary-phase culture of the parental strain was measured in hours and could thus, not be used to explain the instantaneous protective properties of the heated cultures. Lowering the redox potential of the medium by addition of exogenous reductants could bring the time of RpoS induction significantly forward. Even then however, the RpoS-induction times measured were not consistent with the instantaneous protection. Monitoring the growth and RpoS-induction times in S. Typhimurium (pSB367) in mixed cultures with other Gram-negative competitors, it was shown that in the spent medium of stationary-phase cultures of Salmonella and the other Gram-negative organisms tested (E. coli, C. freundii and P. aeruginosa), an extracellular component(s) did exist however, able to produce early RpoS induction in S. Typhimurium (pSB367) in pure culture. Those components were redox sensitive. Oxidation permanently and irreversibly inactivated the extracts from the Enterobacteriaceae. Oxidation followed by reduction could restore the Salmonella growth inhibitory and RpoS-induction abilities of the P. aeruginosa extract. It was shown that in mixed cultures with other Gram-negative organisms, an increase in the redox potential early during growth could allow Salmonella to reach high cell densities and inhibit RpoS induction.